CN106591488A - Nucleic acid combination for detecting Fusobacterium nucleatum in night soil, and applications and kits thereof - Google Patents
Nucleic acid combination for detecting Fusobacterium nucleatum in night soil, and applications and kits thereof Download PDFInfo
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- CN106591488A CN106591488A CN201710101308.9A CN201710101308A CN106591488A CN 106591488 A CN106591488 A CN 106591488A CN 201710101308 A CN201710101308 A CN 201710101308A CN 106591488 A CN106591488 A CN 106591488A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention discloses a nucleic acid combination for detecting Fusobacterium nucleatum in night soil, and applications and kits thereof, and belongs to the technical field of biomedical examination. The nucleic acid combination for detecting Fusobacterium nucleatum in night soil can specifically detect the Fusobacterium nucleatum. The nucleic acid combination is used to prepare a kit for detecting Fusobacterium nucleatum in night soil and a kit for detecting cancer. The kits effectively improve the detection precision, can be conveniently and fast applied to detect the Fusobacterium nucleatum, and have high sensitivity and specificity. A technologic method provided by the invention can be used to conveniently extract Fusobacterium nucleatum genome from the night soil, and has the advantages of quantitative detection of the Fusobacterium nucleatum, simplicity in operation, and facilitation of promotion and application.
Description
Technical field
The present invention relates to biomedical inspection technology field, and more particularly to a kind of core for detecting Fusobacterium nucleatum in feces
Acid combination and its application and test kit.
Background technology
Colorectal cancer (Colorectal cancer, CRC) is common multiple malignant tumor of digestive tract, its sickness rate and
Mortality rate is respectively positioned on the 3rd of whole world common cancer.In China, colorectal cancer incidence rate rises year by year in recent years, estimates
400,000 new cases are there are about every year, rank the 2nd at present in China's alimentary system malignant tumour.Early stage colorectal cancer patients handss
5 years survival rates of art may be up to more than 90%, and in, 5 years survival rates only 10% or so of patients with terminal, the colon cancer of clinical diagnosises is about
80% is middle and advanced stage, and this is one of key factor for causing its mortality rate to be not improved.And effectively early stage straight colon cancer is suffered from
Person can make straight colon cancer morbidity reduce by 60%, and case fatality rate reduces by 80%, thus the early discovery of colorectal cancer, early diagnosiss and
Early treatment is particularly important.
At present, straight colon cancer non-invasive inspection methods are very low for the examination effective force of disease, since it is desired that suffering from
Person collects the test of fecal sample fecal occult blood or Fecal Immunochemical fluoroscopic examination every year, and false positive rate is high, and as a result stability is poor,
Overall examination rate is less than 3%.Although sigmoidoscopy or colonoscopy recall rate are higher, it is the gold of straight colon cancer inspection
Standard, however it is necessary that patient does cleaning INTESTINAL CLEANSING, relies on anesthesia and invasion of privacy, patient produce the feared state of mind, checks
Situations such as bleeding or infection, overall compliance is poor, and examination rate is less than 5%.Therefore, high specific, high sensitivity, compliance
The straight colon cancer early diagnosiss technology of good, non-intrusion type is urgently to be resolved hurrily.
Fusobacterium nucleatum (Fusobacterium nucleatum, Fn) is common Gram-negative without brood cell's anaerobism bar
Bacterium, with the most common in the tartar of oral cavity.October in 2011, two independent research teams confirmed that Fusobacterium nucleatum exists in intestinal
In road, and the abundance of Fusobacterium nucleatum is relevant with colorectal cancer.Harvard Medical School in 2013 confirms tool with CWRU
Core Fusobacterium is opened by inflammation inducing reaction, rush cancer signal path, promotes colorectal cancer propagation.
At present, in terms of being concentrated mainly on oral cavity class disease and common cancer to the application of Fusobacterium nucleatum；Can not enter
Row detection by quantitative, in feces, Fusobacterium nucleatum and its concrete detection method also have no report.
The content of the invention
The first object of the present invention is to provide a kind of Nucleic acid combinations for detecting Fusobacterium nucleatum in feces, the Nucleic acid combinations
Can special, high-sensitivity detection Fusobacterium nucleatum.
The second object of the present invention is to provide the application of above-mentioned Nucleic acid combinations Fusobacterium nucleatum in detection feces.
The third object of the present invention is the application for providing above-mentioned Nucleic acid combinations in the test kit for preparing detection cancer.
The fourth object of the present invention is to provide above-mentioned Nucleic acid combinations in the test kit for preparing detection Fusobacterium nucleatum
Using.
The fifth object of the present invention is to provide a kind of test kit of detection feces Fusobacterium nucleatum, and the test kit can be conveniently
It is quickly applied to detect Fusobacterium nucleatum in feces, it is convenient to operate, beneficial to popularization and application.
The present invention solves its technical problem and employs the following technical solutions to realize.
The present invention provides a kind of Nucleic acid combinations of Fusobacterium nucleatum in detection feces, and Nucleic acid combinations include primer combination and visit
Pin；Primer combination includes the first primer pair, the second primer pair and three-primer pair, the base sequence such as SEQ ID of the first primer pair
Shown in NO.1-2, the base sequence of the second primer pair as shown in SEQ ID NO.3-4, the base sequence such as SEQ of three-primer pair
Shown in IDNO.5-6；Probe includes the first probe, the second probe and the 3rd probe, the base sequence such as SEQ ID of the first probe
Shown in NO.7, as shown in SEQ ID NO.8, the sequence of the 3rd probe is as shown in SEQ ID NO.9 for the sequence of the second probe；Probe
5' ends be marked with fluorophor, probe 3' ends are marked with quenching group, fluorophor be FAM, VIC, TET, JOE, HEX,
One kind in CY3, CY5, ROX, RED610, TEXASRED, RED670, NED, the quenching group be 6-TAMRA, BHQ-1～
3rd, the one kind and in the non-fluorescence quencher of binding molecule ditch.
Further, above-mentioned first primer pair is Fusobacterium nucleatum detection primer, and upstream detection primer includes SEQ IDNO.1
Shown nucleotide sequence, the nucleotides sequence shown in SEQ IDNO.1 are classified as 5 '-TCTCCTTTCAATAAA AGTGGC-3 '；
Fusobacterium nucleatum downstream detector primer includes the nucleotide sequence shown in SEQ IDNO.2, shown in SEQ IDNO.2
Nucleotides sequence is classified as：5’-AAGAAGTAGGATGAAAATCAGTTA-3’；
Further Fusobacterium nucleatum probe includes the nucleotide sequence shown in SEQ IDNO.7, the core shown in SEQ IDNO.7
Nucleotide sequence is 5 '-AGACCGATGCTCTACC-3 '；
Further, the 5' ends of detection probe are marked with fluorophor, and the 3' ends of detection probe are marked with quenching group.
The fluorophor of above-mentioned probe 5' ends labelling is：FAM、VIC、TET、JOE、HEX、CY3、CY5、ROX、RED610、
One kind in TEXASRED, RED670, NED；The quenching group of its 3' ends labelling is：6-TAMRA, BHQ-1～3, and combine point
In the non-fluorescence quencher (Minor Groove Binder nonfluorescent quencher, MGB NFQ) of cunette one
Kind.Preferably, fluorescent marker selects FAM, quencher to select MGB.
Further, the second primer pair and the second probe are internal reference (Internal reference, IR) primer pair and internal reference
Probe；
Internal control primer is to including internal reference upstream primer and internal reference downstream primer, being the 16SrDNA genes for expanding antibacterial；
Shown in the nucleotide sequence SEQ IDNO.3 of internal reference IR forward primer, the nucleotides sequence of SEQ IDNO.3 is classified as 5 '-
CCTGGACGAAGACTGACGCTC-3’；
As shown in SEQ IDNO.4, the nucleotides sequence of SEQ IDNO.4 is classified as the nucleotide sequence of internal reference IR forward primer
5’-CTCAAGGGCACAACCTCCAAG-3’；
Shown in the nucleotide sequence SEQ IDNO.8 of internal reference IR probes, the nucleotides sequence shown in SEQ IDNO.8 is classified as 5 '-
CGAAAGCGTGGGGAGCAAACAGG-3’；
Above-mentioned internal reference IR probes in the fluorophor of the labelling at its 5' end are：FAM、VIC、TET、JOE、HEX、CY3、CY5、
A kind of in ROX, RED610, TEXASRED, RED670, NED, the quenching group of 3' ends labelling is：6-TAMRA, BHQ-1～3, and
The non-fluorescence quencher (Minor Groove Binder nonfluorescent quencher, MGB NFQ) of binding molecule ditch
In one kind.Preferably, fluorescent marker selects NED, quencher to select BHQ-2.
Three-primer to and the 3rd probe be process control crt gene (processing control, PC) primer pair and
Process control crt gene PC probes；Its objective is to monitor the whole detection process from nucleic acid extraction to amplification.
PC primer pairs include PC forward primer and PC downstream primers, are the gene for expanding bacillus subtilises；
Shown in the nucleotide sequence SEQ IDNO.5 of PC forward primer, the nucleotides sequence shown in SEQ IDNO.5 is classified as5’- GATGAATATCCGCATCTACC-3’；
Shown in the nucleotide sequence SEQ IDNO.6 of PC downstream primers, the nucleotides sequence shown in SEQ IDNO.6 is classified as5’- TTTGGCGGATCAGGTTTTTC-3’；
Shown in the nucleotide sequence SEQ IDNO.9 of PC probes, the nucleotides sequence shown in SEQ IDNO.9 is classified as5’- CACGCCATTCAGATTCCGACGGATCT-3’；
The fluorophor of the 5' ends labelling of above-mentioned PC probes is：FAM、VIC、TET、JOE、HEX、CY3、CY5、ROX、
One kind in RED610, TEXASRED, RED670, NED；The quenching group of 3' ends labelling is：6-TAMRA, BHQ-1～3, and knot
Close in the non-fluorescence quencher (Minor Groove Binder nonfluorescent quencher, MGB NFQ) of molecule ditch
One kind.Preferably, fluorophor selects VIC, quenching group to select BHQ-2.
The application of above-mentioned Nucleic acid combinations Fusobacterium nucleatum in detection feces is provided.
Application of the above-mentioned Nucleic acid combinations in the test kit for preparing detection cancer is provided.
Application of the above-mentioned Nucleic acid combinations in detection Fusobacterium nucleatum test kit is prepared is provided.
The present invention also provides a kind of for detecting tool in Fusobacterium nucleatum test kit in feces, including above-mentioned detection feces
The Nucleic acid combinations of core Fusobacterium.
Compared with prior art, the invention has the beneficial effects as follows：The nucleic acid group of the detection Fusobacterium nucleatum that the present invention is provided
Conjunction can specifically detect Fusobacterium nucleatum, prepare the test kit and system of Fusobacterium nucleatum in detection feces using the Nucleic acid combinations
The test kit of standby detection cancer, effectively improves the precision of detection, conveniently can be applied to detect Fusobacterium nucleatum, with compared with
High sensitivity and specificity.The technical method that the present invention is provided, more easily can not only extract Fusobacterium nucleatum from feces
Genome, carries out detection by quantitative to Fusobacterium nucleatum, and simple to operate, beneficial to popularization and application.
Description of the drawings
In order to be illustrated more clearly that the technical scheme of the embodiment of the present invention, below by to be used attached needed for embodiment
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, thus be not construed as it is right
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can be with according to this
A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 is the qPCR result figures of the checking test kit linear analysiss that experimental example of the present invention 2 is provided；
Fig. 2 is the qPCR result figures of the checking test kit minimum detectability that experimental example of the present invention 2 is provided；
Fig. 3 is the qPCR result figures of the checking test kit precision that experimental example of the present invention 2 is provided；
Fig. 4 is the electrophoresis result figure that experimental example of the present invention 2 is provided；
Fig. 5 is the electrophoresis result figure that experimental example of the present invention 2 is provided；
Fig. 6 is the Fusobacterium nucleatum gene expression abundance cartogram that experimental example of the present invention 3 is provided；
Fig. 7 is the Fusobacterium nucleatum expression ROC curve analysis chart that experimental example of the present invention 3 is provided；
Fig. 8 is the qPCR result figures that experimental example of the present invention 3 is provided；
Fig. 9 is the qPCR result figures that experimental example of the present invention 3 is provided；
Figure 10 is the qPCR result figures that experimental example of the present invention 4 is provided；
Figure 11 is the qPCR result figures that experimental example of the present invention 4 is provided.
Specific embodiment
To make purpose, technical scheme and the advantage of the embodiment of the present invention clearer, below will be in the embodiment of the present invention
Technical scheme be clearly and completely described.In embodiment, unreceipted actual conditions person, builds according to normal condition or manufacturer
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, are the conventional product that can pass through that commercially available purchase is obtained
Product.
Above-mentioned Nucleic acid combinations are at Thermo Fischer Scient Inc. (Thermo Fisher Scientific)) company's synthesis.
UNG enzymes, Taq enzyme, dNTPs and Mg2+Purchased from TAKARA；HSTaq enzymes, glycine betaine, DMSO are purchased from Chinese medicines group；
BSA is purchased from Sigma companies.
Example below to the present invention provide a kind of detection feces in Fusobacterium nucleatum Nucleic acid combinations and its application with
Test kit is specifically described.
The Nucleic acid combinations of Fusobacterium nucleatum in a kind of detection feces, Nucleic acid combinations include primer combination and probe；Primer sets
Conjunction includes the first primer pair, the second primer pair and three-primer pair, the base sequence such as SEQ ID NO.1-2 institutes of the first primer pair
Show, the base sequence of the second primer pair as shown in SEQ ID NO.3-4, the base sequence such as SEQ ID NO.5- of three-primer pair
Shown in 6；Probe includes the first probe, the second probe and the 3rd probe, the base sequence such as SEQ ID NO.7 institutes of the first probe
Show, as shown in SEQ ID NO.8, the sequence of the 3rd probe is as shown in SEQ ID NO.9 for the sequence of the second probe；The 5' ends of probe
Be marked with fluorophor, probe 3' ends are marked with quenching group, fluorophor be FAM, VIC, TET, JOE, HEX, CY3, CY5,
One kind in ROX, RED610, TEXASRED, RED670, NED, quenching group are 6-TAMRA, BHQ-1～3, and binding molecule
One kind in the non-fluorescence quencher of ditch.
The fluorescent probe of a specificity is added when PCR is expanded while pair of primers is added, the probe is a few core
Thuja acid, two ends difference one reporter fluorescence group of labelling and a quenching fluorescence group.When probe is complete, reporter group transmitting
Fluorescence signal is quenched group absorptions；When PCR is expanded, probe enzyme action is degraded by 5 ' -3 ' 5 prime excision enzyme activities of Taq enzyme, makes report
Fluorophor is separated with quenching fluorescence group, so as to fluorescence monitoring system can receive fluorescence signal, i.e., often expands a DNA
Chain, just has a fluorescence molecule to be formed, and the accumulation and PCR primer for realizing fluorescence signal forms Complete Synchronization.
Further, the preferred FAM of fluorophor, the preferred MGB of quenching group.
The application of above-mentioned Nucleic acid combinations Fusobacterium nucleatum in detection feces.
Whether combine using above-mentioned primer when containing Fusobacterium nucleatum in detection sample, can fast and accurately area
Separate whether sample contains Fusobacterium nucleatum.
Further, application of the detection primer combination of above-mentioned Fusobacterium nucleatum in detection Fusobacterium nucleatum, which includes：
DNA with sample to be checked adds the primer combination of above-mentioned detection Fusobacterium nucleatum, carries out pcr amplification reaction as template, and PCR expands
Increasing the program reacted is：First stage：50 DEG C of UNG enzymic digestion 10min；Second stage：95 DEG C of denaturations 2min；Phase III：
95 DEG C of 20s, 60 DEG C of 30s, annealing extend 30s, collect FAM/NED/VIC signals, 40 circulations.
In PCR reactions, will anneal and extend two reactions steps and merge, and be conducive to shortening the response time, it is rational to control
The annealing temperature of reaction and and the time, be more beneficial for react carrying out, it is to avoid experimental result is disturbed by non-specific band；40 anti-
Should circulate, be more beneficial for obtaining clear accurately testing result.
Analysis of test results method is as follows：Period Ct needed for the threshold value of setting is reached using fluorescence signal as judgement mark
Standard, contains Fusobacterium nucleatum when genes of interest and internal standard gene are presented during S type amplification curves show sample, is otherwise without tool
Core Fusobacterium, and work as CtFn-CtICWhen >=19, as a result it is judged as feminine gender, works as CtFn-CtICDuring ＜ 19, as a result it is judged as the positive, together
When according to standard curve, the copy number of Fusobacterium nucleatum in every mg stool samples can be calculated, so as in quantitative analyses specimen
The content of Fusobacterium nucleatum.
Application of the above-mentioned Nucleic acid combinations in the test kit for preparing detection cancer.
Further, cancer includes colorectal cancer, excessive risk precancerous polyp.
The cancer of above-mentioned Nucleic acid combinations detection includes but is not limited to colorectal cancer, excessive risk precancerous polyp, can also be which
The detection of the cancer of the type of his correlation.
Application of the primer combination of above-mentioned detection Fusobacterium nucleatum in the test kit for preparing detection Fusobacterium nucleatum.
Using test kit prepared by the primer combination of above-mentioned detection, the result of detection is made more accurately and reliably.
The test kit of Fusobacterium nucleatum in a kind of detection feces, including it is above-mentioned for detecting Fusobacterium nucleatum in feces
Nucleic acid combinations.
Further, in above-mentioned detection feces the test kit of Fusobacterium nucleatum also include sample preservation liquid, nucleic acid extraction liquid,
PCR reactant liquors, positive quality control product, negative quality-control product, internal standard control, one or more in standard substance.
Further, sample preservation liquid includes the TrisHCl and 0.05- of EDTA, 0.5-3mol/L of 0.1-1mol/L
The NaCl of 0.15mol/L.
The EDTA of higher concentration is capable of the effect of effective inhibitory enzyme, so as to prevent the digested degraded of gene, it is to avoid because
The problem of sample, causes detection failure, or causes testing result to go wrong or error.
Further, nucleic acid extraction liquid is washed comprising nucleic acid cleavage liquid, PVPP, E.C. 3.4.21.64, RNase, DNA rinsing liquids and DNA
One or more in de- liquid.
Further, nucleic acid cleavage liquid include 100-500mM TrisHCl, 20-100mMEDTA, 0.4-1M NaCl and
SDS of the volume fraction for 1%-4%.
The TrisHCl of the guanidine hydrochloride and 0-4M of DNA rinsing liquid main components 4-10M；DNA rinsing liquids are in bacterial genomes
During DNA extraction, the impurity such as protein and salt ion are washed away, leave the molecule of pure DNA, play a part of remove impurity.
PVPP (crospolyvinylpyrrolidone), also known as polyvinylpolypyrrolidone, white or near-white is easily flowed with hygroscopicity
Dynamic powder, odorless or micro- smelly, water insoluble, alkali, sour and conventional organic solvent, with very strong expansion character and with multiclass thing
The complexing power of matter.
RNase is a class Cobra venom endonuclease, RNA molecule that can be in degradation reaction system；As RNA molecule is in sequence
There may be similar fragment to DNA, decompose RNA, be avoided that impact of the RNA molecule to reacting.
E.C. 3.4.21.64 is a kind of serine protease of Subtilisin enzyme, is a kind of highly active protein enzyme, for biological sample
The general degraded of protein in product.E.C. 3.4.21.64 can be in bacterium for degrading thalline protein, it is to avoid protein is to the shadow tested
Ring.
DNA eluents need DNA molecular during bacterial genomes DNA extraction, finally the eluting from adsorption column
Out, solution is formed, facilitates further experimental implementation.
Further, PCR reactant liquors include PCR reaction buffers, dUTP, dATP, dCTP, dGTP, thermal starting Taq
Archaeal dna polymerase, UNG enzymes, BSA, glycine betaine, DMSO and Mg2+In one or more.
With reference to embodiments the feature and performance of the present invention are described in further detail.
Embodiment 1
The present embodiment provide it is a kind of detection feces in Fusobacterium nucleatum Nucleic acid combinations, Nucleic acid combinations include primer combination and
Probe；Primer combination includes the first primer pair, the second primer pair and three-primer pair, the base sequence such as SEQ of the first primer pair
Shown in ID NO.1-2, as shown in SEQ ID NO.3-4, the base sequence of three-primer pair is such as the base sequence of the second primer pair
Shown in SEQ IDNO.5-6, the sequence of the first primer pair is as follows：
Forward primer SEQ ID NO.1：
5’-TCTCCTTTCAATAAA AGTGGC-3’；
Downstream primer SEQ ID NO.2：
5’-AAGAAGTAGGATGAAAATCAGTTA-3’；
The sequence of the second primer pair is as follows；
Forward primer SEQ ID NO.3：
5’-CCTGGACGAAGACTGACGCTC-3’
Downstream primer SEQ ID NO.4：
5’-CTCAAGGGCACAACCTCCAAG-3’；
The sequence of three-primer pair is as follows；
Forward primer SEQ ID NO.5：
5’-GATGAATATCCGCATCTACC-3’
Downstream primer SEQ ID NO.6：
5’-TTTGGCGGATCAGGTTTTTC-3’；
Probe includes the first probe, the second probe and the 3rd probe, the sequence of the first probe as shown in SEQ ID NO.7,
, as shown in SEQ ID NO.8, the sequence of the 3rd probe is as shown in SEQ ID NO.9 for the sequence of the second probe；The 5' ends mark of probe
Note has fluorophor, and probe 3' ends are marked with quenching group.
The sequence of probe is as follows：
First probe SEQ ID NO.7：
FAM-5’-AGACCGATGCTCTACC-3’-MGB；
Second probe SEQ ID NO.8：
FAM-5’-CGAAAGCGTGGGGAGCAAACAGG-3’-MGB；
3rd probe SEQ ID NO.9：
FAM-5’-CACGCCATTCAGATTCCGACGGATCT-3’-MGB；
The present invention other embodiments in, fluorophor can also be VIC, TET, JOE, HEX, CY3, CY5, ROX,
One kind in RED610, TEXASRED, RED670, NED, quenching group can also be 6-TAMRA, BHQ-1～3, and combine point
In the non-fluorescence quencher (Minor Groove Binder nonfluorescent quencher, MGBNFQ) of cunette one
Kind.
When in use, the first probe is used cooperatively with the first primer pair, and the second probe is coordinated with the second primer pair to be made
With the 3rd probe is with three-primer to using cooperatively.
The use of the Nucleic acid combinations of Fusobacterium nucleatum in above-mentioned detection feces, concrete grammar are as follows：
With sample DNA to be checked as template, above-mentioned primer pair is added to carry out pcr amplification reaction, PCR reaction systems are 25 μ L；
0.5 μ L, Taq archaeal dna polymerase of template DNA, 0.5 μ L, 10 × buffer buffer, 2.5 μ L, dNTPs (10mM each) 1 μ L,
17.5 μ L of MgSO41 μ L and ddH2O, 0.5 μ L of forward primer, 0.5 μ L of downstream primer.
PCR response procedures are：First stage：50 DEG C of UNG enzymic digestion 10min；Second stage：95 DEG C of denaturations 2min；The
Three stages：95 DEG C of 20s, 60 DEG C of 30s, annealing extend 30s, collect FAM/NED/VIC signals, 40 circulations.
Embodiment 2
The present embodiment provides a kind of test kit of detection Fusobacterium nucleatum, and the test kit includes the core provided such as embodiment 1
Acid combination.
Certainly, this test kit also includes the reagent that can directly enter performing PCR reaction, including PCR reaction buffers, Taq
Archaeal dna polymerase, dNTPs and Mg2+In one or more.
The using method of test kit, pcr amplification reaction system and PCR response procedures that reference implementation example 1 is provided, difference
When being that PCR reacts, the merging temperature for extending of annealing is 65 DEG C.
Embodiment 3
The present embodiment provides the present embodiment and provides a kind of test kit of detection Fusobacterium nucleatum, and the test kit is included as implemented
The detection primer combination of the detection Fusobacterium nucleatum that example 1 is provided.
The present embodiment provide test kit also include sample preservation liquid, sample preservation liquid include 0.1mol/L EDTA,
The NaCl of the TrisHCl and 0.15mol/L of 3mol/L.
Certainly, this test kit also includes the reagent that can directly enter performing PCR reaction, including PCR reaction buffers, Taq
Archaeal dna polymerase, dNTPs and Mg2+In one or more.
The using method of test kit, with reference to following steps：
By the collecting dung of patient in sample preservation liquid, room temperature or 4 DEG C are preserved 2 weeks 1.1 collecting dungs, and -20 DEG C preserve 6
Individual month；In the fecal sample collected to 200mg (or 200uL loose stool), 100 μ L E.C. 3.4.21.64s of addition, the nucleic acid cleavage liquid of 0.5mL,
Fully mix；
1.2 are incubated 10min under the conditions of 70 DEG C；
The PVPP of 1.3 addition 100mg is slightly carried, the impurity such as such as pigment or protein in removal crude extract；
1.4 add 100 μ L of RNase, 37 DEG C of water-baths 1 hour, remove the RNA impurity in solution completely；
1.5 are added to sample in the centrifugal column with adsorbed film, 13000rpm centrifugation 1min, the waste liquid abandoned in collecting pipe；
The 1.6 DNA rinsing liquids 1 for adding 500 μ L, 13000rpm centrifugation 1min, abandon waste liquid；
1.7 addition rinsing liquids 2 are washed, 13000rpm centrifugation 1min, abandon waste liquid, and repetitive operation is once；
1.8 are put into adsorption column in new collecting pipe, and the DNA eluents of 50 μ L of addition (can also be dense according to actual DNA
The addition of the appropriate adjustment DNA eluents of degree), then 13000rpm centrifugations 1min, obtains genome.
The PCR amplification method of test kit, pcr amplification reaction system and PCR response procedures that reference implementation example 1 is provided.
Embodiment 4
The present embodiment provides the present embodiment and provides the present embodiment offer a kind of test kit of detection Fusobacterium nucleatum, the reagent
Box includes the detection primer combination of the Fusobacterium nucleatum provided such as embodiment 1.
The present embodiment provide test kit also include sample preservation liquid, sample preservation liquid include 1mol/L EDTA,
The NaCl of the TrisHCl and 0.05mol/L of 0.5mol/L.
Certainly, this test kit also includes the reagent that can directly enter performing PCR reaction, including PCR reaction buffers, Taq
Archaeal dna polymerase, dNTPs and Mg2+In one or more.
The method that the method reference implementation example 3 that the sample collection of the detection kit that the present embodiment is provided is processed is provided, PCR
Response procedures reference implementation example 1.
Embodiment 5
The present embodiment provides the present embodiment and provides the present embodiment offer a kind of test kit of detection Fusobacterium nucleatum, the reagent
Box includes the detection primer combination of the detection Fusobacterium nucleatum provided such as embodiment 1.
The present embodiment provide test kit also include sample preservation liquid, sample preservation liquid include 0.5mol/L EDTA,
The NaCl of the TrisHCl and 0.1mol/L of 1mol/L；And bacterial lysate, PVPP, E.C. 3.4.21.64, RNase, DNA rinsing liquids
With DNA eluents.
Certain can also include bacterial lysate, PVPP, E.C. 3.4.21.64, RNA in other embodiments of the present invention
At least one in enzyme, DNA rinsing liquids and DNA eluents.
Certainly, this test kit also includes the reagent that can directly enter performing PCR reaction, including PCR reaction buffers, Taq
Archaeal dna polymerase, dNTPs and Mg2+In at least one.
Experimental example 1
This experimental example provides the use of the test kit of embodiment, specifically used as follows：
1st, PCR reaction mixtures
PCR reactant liquors each component constitutes as follows with concentration：Taq archaeal dna polymerase 1.5U, UNG enzyme 0.2U, 0.6 μM of dUTP,
DATP, dCTP, dGTP are each 0.2 μM, the Mg of 2mM2+, Fn forward primer, downstream primer, probe, IR forward primer, downstream primer,
Probe and PC forward primer, downstream primer, probe are 0.2 μM, the glycine betaine of 25mM, 0.1 μ g/ul BSA and 5%
DMSO, supplies H2The μ L of O to 24.
Positive control includes the Fusobacterium nucleatum reference culture for inactivating, and concentration is 107CFU/μL。
Negative control includes non-Fusobacterium nucleatum sample DNA solution.
Standard substance include the DNA fragmentation plasmid of the nucleotide sequence shown in SEQIDNO.10, standard concentration is respectively 1 ×
107Copy/μ L, 1 × 106Copy/μ L, 1 × 105Copy/μ L, 1 × 104Copy/μ L, 1 × 103Copy/μ L.
Process control control includes the bacillus subtilises for inactivating, and concentration is 106CFU/μL。
2nd, fluorescence real-time quantitative PCR reaction and detection
Quantitative fluorescent PCR reaction condition is as shown above：First stage：50 DEG C of UNG enzymic digestion 10min；Second stage：95
DEG C denaturation 2min；Phase III：95 DEG C of 20s, 60 DEG C of 30s, annealing extend 30s, collect FAM/NED/VIC signals, and 40 are followed
Ring.
3rd, result judges
After reaction terminates, deposited as Fusobacterium nucleatum gene according to the amplification curve of sample in each reaction tube and period Ct
In whether basis for estimation.
The first step, the detection signal of the IR and PC of sample is presented S amplification curves and Ct values are less than 38, shows that this is augmented with
Effect；Be less than 38 when PC is presented S amplification curves and Ct values, and IR without amplification or Ct more than or equal to 38 when, show that sample nucleic acid amount is low
In Monitoring lower-cut；When PC is without S amplification curves, show that this amplification is invalid.
Second step, on the premise of amplification effectively：
If sample to be tested produces S types amplification curve and Ct values are less than 38, in the sample, there is Fusobacterium nucleatum gene；
If sample to be tested amplification Ct values are more than or equal to 38, in sample to be tested, there is no Fusobacterium nucleatum gene；
The difference of Ct is expanded according to the Fusobacterium nucleatum gene amplification Ct and 16SrDNA of sample in each reaction tube, as sentencing
The standard of disconnected colorectal cancer and precancerous polyp.
When Fusobacterium nucleatum is normally expanded with internal standard gene, and work as CtFn-CtIRDuring ＞ 19, as a result it is judged as Colon and rectum
Cancer is negative with precancerous polyp, works as CtFn-CtIRWhen≤19, as a result it is judged as that colorectal cancer is positive with precancerous polyp.
Wherein CtFnRepresent Fusobacterium nucleatum gene amplification Ct values, CtIRRepresent 16SrDNA amplification Ct values.
In addition, according to Fn standard curves, the copy number of Fusobacterium nucleatum in every mg stool samples can be calculated, and quantitatively
The content of Fusobacterium nucleatum in analytical specimen.
Experimental example 2
This experimental example provides the Performance Evaluation of the Nucleic acid combinations of Fusobacterium nucleatum in the detection feces in embodiment 2.
Comprise the following steps that：
The preparation of reference material DNA nucleic acid
1.1 as standard substance the plasmid construction comprising Fusobacterium nucleatum genetic fragment
With core Fusobacterium gene order as template, SEQIDNO.10 nucleic acid sequences are synthesized by Wuhan Jin Kairui biotech companies
Row.SEQIDNO.10 is connected to into pUC57 carriers, and check and correction is sequenced.The plasmid gradient dilution of structure is divided into into 1 for concentration ×
107Copy/μ L, 1 × 106Copy/μ L, 1 × 105Copy/μ L, 1 × 104Copy/μ L, 1 × 103The DNA solution of copy/μ L.
1.2 used as plasmid construction of the internal reference comprising 16SrDNA genetic fragments
With 16SrDNA gene orders as template, SEQIDNO.11 nucleic acid sequences are synthesized by Wuhan Jin Kairui biotech companies
Row.SEQIDNO.11 is connected to into pUC57 carriers, and check and correction is sequenced.
1.3 positive controls include the Fusobacterium nucleatum reference culture for inactivating, and concentration is 107CFU/μL。
1.4 negative controls include non-Fusobacterium nucleatum sample DNA solution.
1.5 process control reference substances include the bacillus subtilises for inactivating, and concentration is 106CFU/μL。
For detecting the performance evaluation of Fusobacterium nucleatum gene primer and probe
It is prepared by the reference material of 2.1 variable concentrations
The Fusobacterium nucleatum genetic fragment recombiant plasmid that above-mentioned steps synthesize is carried out quantitatively, and quantification of 1010Copy/μ
L, and gradient dilution, obtain 1 × 107Copy/μ L, 1 × 106Copy/μ L, 21 × 105Copy/μ L, 1 × 104Copy/μ L, 1 ×
103Copy/μ L, 1 × 102Copy/μ L, 1 × 101The variable concentrations gradient dilution liquid such as copy/μ L, as standard substance DNA.
2.2 reaction systems are prepared referring specifically to embodiment 1, and a kind of plasmids of gradient dilution of 1 μ L are added in each reaction tube
Masterplate.
2.3 fluorescence real-time quantitative PCRs are reacted with detection referring specifically to embodiment 1.
As a result judge
As a result as shown in figure 1,6 curves in figure represent that Fusobacterium nucleatum plasmid is diluted to 10 by ten times respectively7、106、
105、104、103、102Individual copy, amplification have an obvious S types amplification curve, and reaction system is 107-102Between it is linear
Distribution, R2≥0.99.Judged according to amplification curve Ct values, concentration has good linear relationship with amplification cycles number Ct results.
As a result as shown in Fig. 2 IR plasmids are diluted to 10 successively6、105、104、103、102、101The Concentraton gradient of individual copy
Solution, amplification have an obvious S types amplification curve, and reaction system is 106-101Between be linearly distributed, minimum detection 1 is copied
The template of shellfish/μ L.These results suggest that the primer, probe have higher amplification sensitivity, and in minimum detectability experiment
Show the template of reaction system 1 copy/μ L of minimum detection.
As a result as shown in figure 3, choosing Fn plasmid concentrations by above-mentioned linear relationship selects 1 × 107Copy/μ L, 1 × 104Copy
Shellfish/μ L, 1 × 102Three Concentration Testings such as copy/μ L prepare reference material solution, and amplification has obvious S types amplification bent
Line, is repeated 10 times per concentration, and CV ＜ 5% these results suggest that the primer, probe amplification precision are good.
As shown in Figure 4 and Figure 5, (in figure, M represents DNA Marker to electrophoresis result：DL500,1-8 represent 8 experiment samples
This), the test kit that the present embodiment is provided can amplify the purpose band of 128bp sizes, and band is clear, homogeneous without miscellaneous
Band；Illustrate that test kit can quickly and accurately amplify band, and with higher specificity and sensitivity.
Experimental example 3
This experimental example using embodiment 4 provide detection feces in Fusobacterium nucleatum test kit to colorectal cancer patients excrement
Just the detection of the Fusobacterium nucleatum in sample；And the ROC curve analysis to Fusobacterium nucleatum in colorectal cancer patients feces.
Colorectal cancer patients 82 during 1.1 samples preparation collection 2014-2016, normal healthy controls 148.Wherein Colon and rectum
Cancer patient age scope between 34-76 year, female patient 37, male patient 45.The concrete details of case is shown in Table 1 to table
5。
The basic condition of 1 case of table
| Total case | 82 |
| Male patient | 45 |
| Female patient | 37 |
| The range of age | 34-76 |
| Mean age | 56.5 |
The different type distribution of 2 colon cancer of table
3 cancer pathology of table is classified and breaks up situation
| Cancer pathology is classified | Case load | Tumor differentiation degree | Case load |
| Adenocarcinoma | 37 | Differentiated | 17 |
| Mucinous adenocarcinoma | 25 | Middle differentiation | 30 |
| Signet-ring cell carcinoma | 20 | Low differentiation | 35 |
4 tumor-infiltrated situation of table
| Tumor-infiltrated degree | Case load | Lymph node staging | Case load |
| Tis | 18 | N0 | 17 |
| T1 | 20 | N1 | 19 |
| T2 | 15 | N2 | 43 |
| T3 | 16 | NX | 3 |
| T4a | 13 |
5 pathological staging of table and metastasis
From table 1 to table 5 as can be seen that tumor patient cover ascending colon cancer, Transverse Colonic Carcinoma, carcinoma of descending colon, sigmoid colon with
Rectal cancer etc., tumor mean size 3.4cm, pathological classification include adenocarcinoma, mucinous adenocarcinoma, signet-ring cell carcinoma, and pathological staging is included
0- IV etc., specifying information are shown in Table 1 colorectal cancer information table.Normal healthy controls the range of age 34-67 year, is that intestinal mirror makes a definite diagnosis non-Colon and rectum
Cancer or polyp patient.
By the collecting dung of patient in sample preservation liquid, room temperature or 4 DEG C are preserved 2 weeks 1.2 collecting dungs, and -20 DEG C preserve 6
Individual month.
1.3 feces genomes are extracted to 200mg (or 200uL loose stool) in the fecal sample collected, and add the nucleic acid of 0.5mL
Lysate, fully mixes；70 DEG C of incubation 10min；Add 50mg PVPP slightly carried, remove crude extract in such as pigment or egg
The impurity such as white matter；100 μ L of 100 μ L of E.C. 3.4.21.64 and RNase are sequentially added, the protein impurities and RNA in solution are removed completely
Impurity；Sample is added in the centrifugal column with adsorbed film, the DNA rinsing liquids washing of 500 μ L, then 13000rpm centrifugations is added
1min, repetitive operation is once；The DNA eluents of 50 μ L are added (can also suitably to adjust DNA eluting according to actual DNA concentration
The addition of liquid), then 13000rpm centrifugations 1min, obtains genome.
1.4PCR amplified reaction pcr amplification reaction systems and amplification program are with specific reference to embodiment 1.
The data processing method of Fusobacterium nucleatum expression is Δ Ct methods.Circulations of the Ct for needed for when reaction reaches threshold value
Number, expression equation 2 of every kind of Fusobacterium nucleatum relative to standard internal reference-ΔCtRepresent, wherein Δ Ct=Ct testing samples-Ct
Internal reference.In feces of the present invention, 16SrDNA is used as internal reference.Data analysiss are carried out using SPSS20.0 softwares, and data presentation technique is
Mean value ± standard error (means ± S.E.M), is compared between group and is checked using t, P<0.05 thinks there is significant difference.
As shown in fig. 6, the Fusobacterium nucleatum relative expression quantity in colorectal cancer patients feces is all remarkably higher than normal control
Group (14.8 ± 6.4vs23.3 ± 4.3), difference have statistical significance (p<0.05).
The specificity and susceptiveness of Fusobacterium nucleatum is calculated by area under ROC curve and the cut-off values selected.
Testing result is as shown in Fig. 7 and Biao 6.
6 Fusobacterium nucleatum detection data of table
| Mark | Threshold value | Sensitivity | Specificity | AUC |
| Fusobacterium nucleatum | 524288 | 76% | 89% | 0.9 |
As Δ Ct=19, under the ROC curve of Fusobacterium nucleatum detection colorectal cancer, area is maximum, and AUC=0.9, this
When corresponding sensitivity and specificity be respectively 76% and 89%.
Experimental example 4
The test kit of Fusobacterium nucleatum in the detection feces that embodiment 3 is provided is provided in this experimental example in detection Colon and rectum
Cancer, the sensitivity of precancerous polyp and specificity.
77 Patients with Colorectal Cancer samples are set in the present embodiment, including break up in 26 adenocarcinoma, 13 well-differentiated adenocarcinomas,
15 part body of gland intraepithelial neoplasias, 23 poorly differentiated adenocarcinomas and 34 other tumor samples, including 7 stomach tubuloses
Adenocarcinoma, 12 gastric gland scale cancer, 10 malignant change of benign gastric ulcer, 5 pancreas small cell carcinomas and 102 normal healthy controls, internal reference IR are antibacterial
16SrDNA, enter performing PCR reaction amplification, PCR reaction amplification systems and response procedures reference implementation example 1.
As a result show, for middle differentiation adenocarcinoma, its detection sensitivity are 84.3%, specificity is 88.2%.
For well-differentiated adenocarcinoma, its detection sensitivity is 73.9%, and specificity is 85.3%.
For body of gland intraepithelial neoplasia, its detection sensitivity is 49.6%, and specificity is 89.3%.
For poorly differentiated adenocarcinoma, its detection sensitivity is 83.5%, and specificity is 87.7%.
As shown in Figure 10, Figure 11, amplification curves of the wherein Figure 10 for Fusobacterium nucleatum, Figure 11 are the second primer to partial results
To amplification curve.In figure, 12 Patients with Colorectal Cancer sample △ Ct values are all within 19 amplification cycles, other tumor samples
With in blank except the △ Ct values for having a pancreas small cell carcinoma and a gastric gland scale cancer less than 19 circulation in addition to, remaining sample
△ Ct values are all higher than 19 circulations.
Test it can be seen that the primer pair and probe pin specificity of the Nucleic acid combinations for providing of the invention are very strong, clever from above-mentioned
Sensitivity is very high.
In sum, the knowledge that the Nucleic acid combinations of the Fusobacterium nucleatum in detection feces provided in an embodiment of the present invention can be special
The genome sequence of other Fusobacterium nucleatum, rapidly and efficiently detect Fusobacterium nucleatum, as a result accurately and reliably；Using the nucleic acid group
Detection kit of the combination system for Fusobacterium nucleatum, can conveniently help to detect the Fusobacterium nucleatum in sample, detection
As a result accurately, it is easy to use, be conducive to practical application and popularization.
Embodiments described above is a part of embodiment of the invention, rather than the embodiment of whole.The reality of the present invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of the selected enforcement of the present invention
Example.Based on the embodiment in the present invention, what those of ordinary skill in the art were obtained under the premise of creative work is not made
Every other embodiment, belongs to the scope of protection of the invention.
SEQUENCE LISTING
<110>The gloomy Life Science company limited of Wuhan Amy
<120>The Nucleic acid combinations of Fusobacterium nucleatum and its application and test kit in a kind of detection feces
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> Fusobacterium nucleatum
<400> 1
tctcctttca ataaaagtgg c 21
<210> 2
<211> 24
<212> DNA
<213> Fusobacterium nucleatum
<400> 2
aagaagtagg atgaaaatca gtta 24
<210> 3
<211> 21
<212> DNA
<213> Fusobacterium nucleatum
<400> 3
cctggacgaa gactgacgct c 21
<210> 4
<211> 21
<212> DNA
<213> Fusobacterium nucleatum
<400> 4
ctcaagggca caacctccaa g 21
<210> 5
<211> 20
<212> DNA
<213> Fusobacterium nucleatum
<400> 5
gatgaatatc cgcatctacc 20
<210> 6
<211> 20
<212> DNA
<213> Fusobacterium nucleatum
<400> 6
tttggcggat caggtttttc 20
<210> 7
<211> 16
<212> DNA
<213> Fusobacterium nucleatum
<400> 7
agaccgatgc tctacc 16
<210> 8
<211> 23
<212> DNA
<213> Fusobacterium nucleatum
<400> 8
cgaaagcgtg gggagcaaac agg 23
<210> 9
<211> 26
<212> DNA
<213> Fusobacterium nucleatum
<400> 9
cacgccattc agattccgac ggatct 26
Claims (10)
1. a kind of Nucleic acid combinations for detecting Fusobacterium nucleatum in feces, it is characterised in that the Nucleic acid combinations include primer
Combination and probe；The primer combination includes the first primer pair, the second primer pair and three-primer pair, first primer pair
, as shown in SEQ ID NO.1-2, the base sequence of second primer pair is as shown in SEQ ID NO.3-4, described for base sequence
The base sequence of three-primer pair is as shown in SEQ ID NO.5-6；The probe includes the first probe, the second probe and the 3rd spy
Pin；The base sequence of first probe as shown in SEQ ID NO.7, the sequence such as SEQ ID NO.8 institutes of second probe
Show, the sequence of the 3rd probe is as shown in SEQ ID NO.9；The 5' ends of the probe are marked with fluorophor, the probe
3' ends are marked with quenching group, the fluorophor be FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610,
One kind in TEXASRED, RED670, NED, the quenching group is 6-TAMRA, BHQ-1～3, and binding molecule ditch is non-glimmering
One kind in optical quenching agent.
2. the application of Nucleic acid combinations as claimed in claim 1 Fusobacterium nucleatum in detection feces.
3. application of the Nucleic acid combinations as claimed in claim 1 in the test kit for preparing cancer early detection.
4. application according to claim 3, it is characterised in that the cancer includes colorectal cancer, excessive risk precancerous polyp.
5. application of the Nucleic acid combinations as claimed in claim 1 in detection Fusobacterium nucleatum test kit is prepared.
6. in a kind of detection feces Fusobacterium nucleatum test kit, it is characterised in that be used to examine including as claimed in claim 1
Survey the Nucleic acid combinations of Fusobacterium nucleatum in feces.
7. test kit as claimed in claim 6, it is characterised in that the test kit also includes sample preservation liquid, nucleic acid extraction
Liquid, PCR reactant liquors, positive quality control product, negative quality-control product, internal standard control, one or more in standard substance.
8. test kit as claimed in claim 7, it is characterised in that the sample preservation liquid include 0.1-1mol/L EDTA,
The NaCl of the TrisHCl and 0.05-0.15mol/L of 0.5-3mol/L.
9. test kit as claimed in claim 7, it is characterised in that the nucleic acid extraction liquid includes nucleic acid cleavage liquid, PVPP, egg
One or more in white enzyme K, RNase, DNA rinsing liquids and DNA eluents.
10. test kit as claimed in claim 9, it is characterised in that described nucleic acid cleavage liquid includes 100-500mM
TrisHCl, 20-100mM EDTA, 0.4-1M NaCl and the SDS solution that volume fraction is 1%-4%.
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